The ideal tool for eliminating Plasmodium falciparum (Pf), the causative agent of 99% of all malaria deaths, is a highly effective vaccine that entirely prevents blood stage infection and thereby prevents all disease and transmission. Sanaria's goal is to develop and commercialize a Pf sporozoite (SPZ) vaccine that prevents Pf blood stage infection in > 80% of recipients. Sanaria developed a vaccine composed of attenuated, purified, aseptic, cryopreserved PfSPZ, the PfSPZ Vaccine based on the chloroquine-sensitive NF54 strain of Pf. It is safe and well-tolerated when administered to human volunteers by intradermal (ID), subcutaneous (SC) or intravenous (IV) injection (1). It is highly immunogenic and conferred high level protection (100%) against homologous controlled human malaria infection (CHMI) when administered IV (R. Seder et al., in prep.). Roestenberg and co-workers (2) have independently demonstrated high level (100%), long-lasting (28 months) protection with non-attenuated NF54 PfSPZ administered to chloroquine-treated volunteers by the bites of mosquitoes, an approach referred to as chemoprophylaxis with sporozoites (CPS). We have achieved successful infection of volunteers with ID administration of PfSPZ (called PfSPZ Challenge) (3), and recently infected 100% of volunteers (9/9) with IV administration of 3,200 PfSPZ (Mordmueller, in prep.). These findings with PfSPZ Challenge establish the means to rapidly translate CPS into a practical vaccine approach, termed PfSPZ-CVac, in which PfSPZ Challenge is administered with chloroquine chemoprophylaxis. The first clinical trial of PfSPZ-CVac is now in progress. While the limited available evidence suggests that whole PfSPZ immunization can induce protection against homologous and heterologous CHMI challenge, we assume that a multivalent vaccine composed of at least 3 Pf strains of diverse geographic origin will best insure worldwide protection. Our goal in this project is to identify chloroquine sensitiv strains of Pf that can be used: 1) in CHMI to assess the broad-spectrum efficacy of PfSPZ-based vaccines and 2) if there is a need, to include these strains as multivalent vaccine components to achieve broad efficacy. To accomplish this we will adapt to continuous culture at least 2 chloroquine sensitive strains of Pf from Africa and Asia that exhibit asexual growth and sexual stage and sporogonic development characteristics suitable for Sanaria's PfSPZ manufacturing process. During the past 30 yrs, only a few parasite strains have been established that are capable of producing suitably abundant PfSPZ from cultured gametocytes. Achieving our aims will require Sanaria's unique, industrialized approach to PfSPZ production, including systematic screening of multiple chloroquine sensitive parasite isolates in Sanaria's R&D facility where gametocyte culture, mosquito infection, and PfSPZ extraction and quantitative analysis is carried out continuously. The ability to test heterologous protective efficacy and initiate multivalent vaccine manufacturing using heterologous Pf strains could reduce the time to market for PfSPZ vaccines by at least 2 years, with cost savings of tens to hundreds of millions of dollars.